REACTION COLUMNS FOR SIMULTANEOUS MULTIPLE MEASUREMENT AND METHOD

Abstract: Reaction column for simultaneous multiple measurement via affinity assay with at least one substrate bed, comprising at least one affinity reactive component being bound to each substrate bed, and the volume of each bed being up to 600 mu l.

Description: REACTION COLUMNS FOR SIMULTANEOUS MULTIPLE MEASUREMENT AND METHOD. The present invention relates to reaction columns for measure¬ ment via affinity assay with at least one substrate bed. Affinity chromatography is a technology which is broadly ap¬ plied in preparative purification of bio-molecules. Therein, advantage is taken of a specific interaction between the mole¬ cule to be determined and a complementary binding partner. In the general practice of such methods, a sample containing the bio-molecule to be purified is applied to a chromatographic column, which contains one of the mutually complementary bin¬ ding partners bound to a solid substrate. Examples of pairs of complementary binding partners are enzymes and their substra¬ tes, antibodies and antigens or haptens and mutually comple¬ mentary DNA or RNA single chains. In immunoaffinity chromatography for quantification use is made of the interaction between an antigen or a hapten and the complementary antibody. The detection of more than one component in a test sample is still a big problem because usually the separate detection of each compound in a separate assay is necessary. Such detection methods have been preferred since they allow for stringent quality assurance determinations to be performed for each ana- lyte to be tested. In the meantime, laboratories are faced with the problem of providing increasing amounts of tests in a timely manner while attempting to keep costs down. For example, the testing requi¬ rements of blood banks have increased dramatically due to the addition of Human T-Leukemia Virus Type 1 (HTLV-1) , Human Immunodeficiencies Virus 1 and 2 (HIV-1 and HIV-2) and Hepati¬ tis C Virus (HCV) to the panel of agents tested in these labo¬ ratories on donor blood for the presence of or exposure to these agents. Therefore, there is a need for a fast simultaneous analysis of several components or parameters from only one sample with small sample volumes and within a short time. Further such an analysis should also be possible when distur¬ bing components are present which happens often in the field of environmental and nutritional analysis as well as in medi¬ cal diagnostics. Especially in medicine short time analysis, small sample volumes, selectivity, precision and automatical performance are needed features. With the already known me¬ thods of immunologic analysis such features can not be fully realized. The newer state of the art proposes to solve this problem by several methods which are mostly inefficient. At the moment, their are mainly two different kinds of state of the art. The first group describes modified ELISA plate test which on one side can be used from multiple measurements but which have on the other side several draw backs. The other group are test methods which are based on column chromatogra¬ phy. Such methods are only available for single analysis me¬ thod and not for multi-measurements. Thus, the WO-A-91/13354 describes a flow immunosensor method and an apparatus for performing this method. The method of detecting a target moiety comprises the steps of providing an antibody specific to the target, saturating the antibodies binding sites with a labelled form of the target, flowing a liquid containing the target past the antibody, allowing the target to displace the labelled antigen and detecting the displaced, labelled antigen. The drawback of this state of the art is, that it can not be applied for multi-measurement. It is only a competitive me¬ thod. A further drawback is that the displacement of the la¬ belled antigen occurs under non-equilibrium conditions. The non-equilibrium reaction is strongly dependant from the flow rate, which is controlled by a pump and from the temperature. Thus, wrong signals can be generated by high temperatures or organic solvents. Therefore, a second control column with non¬ specific antigens is necessary to give a control signal so that it can be seen whether wrong signals are generated in the test column. With the method of this state of the art only qualitative analysis and single measurement is possible. After a signal is generated the exchange area in the column is re¬ placed or regenerated and it is pointed out that a distinct positive signal is still generated when a 250 ng sample is added after previous additions of antigens. From this state¬ ments can be seen that the method of the state of the art gives no quantitative determination of components. The rela¬ tion between the value of measurement and the concentration is not linear. The detector in the flow through step is, therefo¬ re, only adjusted to one certain analyte. The problem in sin¬ gle measurement makes a multi-measurement impossible. The EP-A-0473 065 discloses an assay to simultaneously detect the presence of one or more analytes in a test sample. The analytes are captured on different solid phases and the pre¬ sence of one or more analyte is determined by detecting a signal generated by analyte-specific indicator reagents. This assay is a modified ELISA plate test which needs long incuba¬ tion times and is difficult in temperature control. WO-A-92/12255 describes assays for the determination of mul¬ tiple analytes which may be present in a test sample by using different short-lived and long-lived chemiluminescent labels and integrating the generated chemiluminescence signal and time discriminating the short-lived and the long-lived com¬ ponents of the signal generated. Also provided are assay kits which contain these short-lived and long-lived chemilumines¬ cent compounds. This method also describes an ELISA plate test with long incubation time and difficult temperature control. Finally, the DE-A-42 08 732 and the DE-C-41 26 436 of the applicant describe reaction columns for solid phase immuno- analysis and a method to determine components detectable by immunoreactions. These columns are easy to handle, work in flow through procedure, need short reaction times and give quantitative reactions. After a basic standardization of the respective lot of columns, further prior calibration or rege¬ neration are not necessary nor a reference standard sample measurement. With such a column all known reactions (like ELISA plate or immunoblotting) are easier and faster or more precisely to perform and can be easily automated. But with the known reac¬ tion columns only measurement of one component is possible. Therefore, it was the object of the invention to provide a reaction column for simultaneous multiple measurement which allows the rapid performance of simultaneous qualitative or quantitative analysis of several components or parameters with solid phase affinity analysis and has the same advantages as the columns described above. The object of the present invention is solved by a reaction column for simultaneous multiple measurement via affinity assay with at least one substrate bed comprising at least one affinity reactive component being bound to each substrate bed and the volume of each bed being up to 600 μl. Preferably, the substrate bed is a solid porous material or a gel. In a preferred embodiment the substrate bed is positioned between two porous separating devices. The reaction column can contain components for compound-specific affinity reactions or components for group-specific affinity reactions or both. These compounds can be immunologically reactive. To each sub¬ strate bed there can be applied at least one reactive compo¬ nent for group- and/or compound-specific reactions. The reaction column according to the invention is prepared with a substrate bed and a reactive component for the affinity reaction in confected and standardized form, allowing imme¬ diate use of the column without any calibration steps or refe¬ rence measurements. The confection has to happen after the quantitative reaction. The reactive components for group-specific affinity reactions and for compound specific affinity reactions have preferably such a loading density that the compounds are bound quantita¬ tively to the substrate bed during the controlled flow of the liquid sample before it leaves the column. The quantitatively bound compounds are the compounds to be determined, or a group containing them or affinity reactive secondary binding partners for the compounds which are applied to the column in any sensible order and number of steps. In a preferred embodiment the reactive components on the sub¬ strate bed are protein A, protein B, protein G, protein M, an immunoglobulin or a group of immunoglobulins, an antigen or a group of antigens. Other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member, for exam¬ ple, an analyte-analog. Immunoreactive specific binding mem¬ bers include further antigens, antigen fragments; antibodies and antibody fragments, monoclonal and polyclonal; and com¬ plexes thereof, including those formed by recombinant DNA me¬ thods. Also in the antigen and the group of antigens hapten is included. The substrate material can be selected from the group contai¬ ning polymeric sugars, plastics, modified plastic organic or inorganic support materials, porous metals, metal oxides, and alloys, glasses, silicates, or ceramics. As polymeric sugar agarose can be used. The reactive component can be bound to the substrate by cova- lent or any kind of sorptive binding. As upper and lower separating devices of one or several sub¬ strate beds porous frits or membranes can be used, if neces¬ sary. The frits have a porosity of 0.2 μm to 100 μm and are made of plastic material, e.g. polyethylene, metal or glass. As plastic material polyethylene or teflon can be used and as metal porous aluminum or stainless steel. The column material further can be selected from the group containing plastics, metals, and natural materials, for example, polyethylene, polypropylene and/or polystyrene as plastic material. The opposite ends of the reaction columns can be shaped to allow male-female connections for in series connection of se¬ veral reaction columns. The substrate bed has preferably a volume of 30 to 50 μl. In special cases, where compounds are present which disturb the affinity reaction the sequence of substrate bed can be arranged in such a manner that a compound which is disturbing the affinity reaction of another compound, is bound quantita¬ tively in an upper substrate bed, while the affinity reaction of the other compound takes place in a lower substrate bed. In a preferred embodiment, additional beds or zones can be in¬ tegrated above or below the measuring substrate beds, such as filtration devices or zones for sample purification or treat¬ ment, additional reaction zones for preparation of the affini¬ ty binding reaction, or control zones for control of sample application, sample flow, and of applied chemicals. The invention further relates to a method for the determina¬ tion of compounds being determinable via an affinity reaction, wherein a sample to be analyzed is applied to a reaction co¬ lumn described above, and the compounds to be analyzed or a group containing them are bound quantitatively by the comple¬ mentary reactive component in the reaction column, whereby the components to be analyzed are determined thereafter by known determination methods after known affinity assay procedures in the reaction column. An affinity reaction may be used before to bind the complemen¬ tary reactive component itself to the column, loaded with a complementary reactive component to this complementary reac¬ tive component (see example 3) . This allows more flexible use of the column, i.e. for different assays and for assay deve¬ lopment, including hiding of the real application of the final user to the producer of the column. Such methods can be secondary affinity reaction in the reac¬ tion column for labelling and/or amplification and determi¬ nation in the column, or determination after elution. Furthermore, affinity reaction of labelled compounds competing with compounds to be determined, and determination directly in the liquid leaving the column, directly in the column, or after elution is possible. As labels fluorescence and/or enzyme labels are used. In a specific embodiment, the group bound by the group-speci¬ fic component is the group of immunoglobulines G and/or M. In a further embodiment, a solution of similarly labelled antigens with one enzyme or fluorescence label for all com¬ plementary antigens can be applied for the qualitative deter¬ mination of the presence of any of special immunoglobulines G and/or M. For quantitative or differentiated qualitative de¬ termination of special immunoglobulines G and/or M a solution of differently labelled antigens can be applied and the anti¬ gens are bound via affinity reactions in direct relation to the amount of bound corresponding immunoglobulines G and/or M. The different antigen labels are different enzyme or fluores¬ cence labels for each complementary antigen. Furthermore, control affinity reactions in respective substra¬ te beds can be used to control the application and the flow of the sample liquid and the quality of the applied reagents. In a preferred embodiment, the reaction column can directly be connected to and remaining at the container of the liquid to be tested, or a blood container especially directly to its filling device for the purpose of safe sampling and test ap¬ pointment. The test result can thus be seen in the moment of usage. Because of the basic standardization and confection of the columns, the method can be performed without calibration and without regeneration of the substrate bed and of the comple¬ mentary reactive component. Additionally, small suction or pressure forces can be applied to the reaction column after sample application for more rapid performance. Further a han¬ dling automate can be used for automated sampling, sample ap¬ plication, and/or measurement. With a reaction column and the method of the invention, it is possible to determine qualitatively or quantitatively, for example, all or specific immunoglobulines of the classes IgG, IgM, IgA and/or IgE in body fluids. Furthermore, a rapid performance of an immuno assay in the preferred flow through procedure is possible without taking notice of an applied exact temperature control, temperature and time dependance, which is easy to handle, which needs not only no precise incubation times, but no incubation times at all, and which is ready to be used without prior calibration and regeneration and without simultaneous reference standard sample measurement. In medical diagnostics it is often important to receive a fast and easy qualitative yes/no response for one or several para¬ meters without any further differentiation, but at very low concentrations, i.e. the assay must be as precise as a quan¬ titative one. The question is, if one of several infections (HIV, HBV, HCV etc.) is present, one of several drugs, one of several doping agents or one of several allergens causing allergy. With the reaction column according to the invention, such a qualitative and also quantitative analysis is possible in an easy and efficient way. The presented method using the reaction column according to the invention is much simpler than common procedure and allows a rapid quantitative and qualitative determination. This is enabled partly because the determination of the component to be determined does not require a preceding calibration or regeneration of the used substrate in the column and because no series reference measurements of standard solutions are necessary. The easy handling is a consequence of the possibility, to apply samples and other solutions by usual laboratory equip¬ ment or with a pipetting automate. The present procedure can be performed rapidly under the force of gravitation without application of high pressure to the reaction column after sample application. But low suction or pressure forces can be used for quicker performance, for exam¬ ple, by centrifugation or pumping. This may be important for substrate beds with large flow resistance to keep the time for flow-through and thus the test time low. In the method of the invention the sample to be analyzed is applied to a reaction column with several different immunolo- gically reactive components . The components to be determined are retained by the complementary immunologically reactive component (s) . Thereafter, the compounds are quantitatively or qualitatively analyzed as described in DE-C-41 26 436 via fluorescence and/or an enzyme colour reaction in the substrate bed in the liquid leaving the column after solution applica¬ tion, or after elution. For quantitative analysis different labels can be used at the antigen or antibody or further com¬ plementary binding partners. If the concentration of the compounds to be analyzed is very low, sample enrichment can be performed by applying larger amounts of sample liquid, if available. Besides, for the de¬ termination of low concentrations of specific antibodies, instead of loading the column with complementary antigens of low molecular weight with corresponding problems in the three- dimensional structure of the substrate bed, the reaction co¬ lumn is loaded with an antibody or a group of antibodies com¬ plementary to the respective class or classes of antibodies (e.g. to human IgG and/or IgM and/or IgA) or a respective group-specific reactive component or respective group-specific reactive components such as protein A or G. With such a column it is possible to retain a whole group of components . In a further step, a solution with an antigen of the component or with antigens of the components to be analyzed is applied to the column. This method has the advantage, that it has a higher sensibili¬ ty, can be better adjusted and that one type of column can be used for different multi- or single tests. The specific kind of the test is determined by the antigens applied afterwards to the column. This is more effective from economical reasons. Further several separated substrate bed volumina with diffe¬ rent immunological and other reactive components can be used in one or more reaction vessel one upon another. Thus, dif¬ ferent separating devices are used in the column, for example, membranes or frits or several substrate beds with different materials which are positioned between porous separating devi¬ ces. The use of various substrate beds allows to perform different methods which are described in the following. For a sample with several components to be analyzed, a reac¬ tion column is used with the number of separated substrate beds corresponding to the number of components. These substra¬ te beds are loaded with one or several complementary reactive partners for the compounds or group of compounds to be ana¬ lyzed. During the sample flow the corresponding compound is retained in the respective substrate bed and, for example, indicated by colour reaction. Disturbing similar compounds with high affinity binding constants are removed quantitati¬ vely and are determined, if wanted, in the upper substrate beds. In the lower bed(s), the determination of the com¬ pound(s) with lower affinity constant (s) follow(s) . If the used gel bed, useful for one or some of the needed reactive components, is incompatible to one or some other nee¬ ded reactive components, the reactions are performed in dif¬ ferent suitable gel beds within one column. Also a simulta¬ neous test of small and large components (antibodies/antigens, bacteria or viruses) is possible, if an upper gel bed is as¬ signed for the larger components, as described in DE-A- 42 08 732. If important tests are performed, like infection test in medi¬ cine or toxicity tests in the analysis of nutrition, control fields are introduced in the upper and lower part of the co¬ lumn. The upper negative control proves the exact flow of the sample the lower positive control proves, if the reactive com¬ pounds were loaded on the column in the right way, and if the sample applied to the column and the used reagents are in good quality. After having performed the test, the reaction column remains at the tested sample, which should be interesting for blood banks. the test column can be connected before the test with a blood container, for example, with a T-piece between the container and the vene of the blood giver. Then the test is performed after taking of the blood sample with the remaining blood in the connected tube. The test vessel remains at the blood container, so that the test result is recognizable at any time without any documentation error. Figure 1 shows the reaction column according to the invention. The column has n substrate beds and is used for simultaneous multiple measurement. 1,2,3, ... ,n represent the different sub¬ strate beds with the attached reaction compounds. 11,12,13,14, 15,...,n' are seperating devices. Figure 2 shows the standard curve for the simultaneous detec¬ tion of tetanus and diphtheria antibody titers (see table 2 and example 2) . Figure 3 shows the standard curve for antibodies in slaughter cattle (see table 3 and example 3) . The following examples show preferred embodiments of the in¬ vention. Examples Preparation of Columns wi th Gel Beds separated by Fri ts Commercial cyanobromide-activated cross-linked agarose (Sepha- rose 4b, Pharmacia, Uppsala, Sweden) was swollen in 0.001 M HCl for 15 minutes, and washed on a glass frit with 0.001 M HCl. 1 ml of a solution of 2 mg of the respective antibody (or antibodies) , antigen (or antigens) , or of protein G in coup¬ ling buffer (0.1 M NaCO 3 -0.5 M NaCl-pH 8.3) and 1 ml of the suspended agarose were mixed in an overhead mixer for 2 hours at room temperature to give an agarose with homogeneously coupled antibody. The degree of antibody or antigen coupling, i.e. amount of bound to amount of added antibody resp. anti¬ gen, was determined by the protein UV absorption of the sus¬ pension at 280 nm (with a Kontron Uvikon photometer) to be nearly 100 %. Afterwards, the antibody- resp. antigen- resp. protein G-coupled suspension was rebuffered on a glass frit with blocking buffer (0.1 M Tris-0.5 M NaCl-pH 8.0) . After blocking for 2 hours at room temperature in the overhead mi¬ xer, the suspension was brought back to a frit and washed four times consecutively by 3 buffers (0.1 M acetic acid-0.5 M NaCl-pH 4.0/coupling buffer/blocking buffer). To each of the polypropylene columns as shown in figure 1, fitted at the lower ends with polypropylene frits 11 with a porosity of 2 μm, 60 μl of the suspended agarose are carefully added. The suspensions are then overlayεred carefully with 500 μl washing buffer each (0.01 M Tris-0.15 M NaCl-0.005 % Tween 20-pH 8.0) . Sedimentation for 1 hour gave final agarose gel substrate beds of about 30 μl. Therefore, the loading density was about 60 μg antibody, antigen, or protein G per 30 μl final agarose bed, corresponding to the coupling degree of about 100 %. The an¬ tibody binding capacities for 30 μl sepharose beds were about 20 μg for antibody-loaded resp. 200 μg for protein G-loaded substrate beds. The beds were closed carefully with upper second frits 12. In the case of multi-field columns, this first procedure gave the respective lowest substrate bed 1, and was then carefully repeated for each of the further neces¬ sary substrate beds above the first one (beds 2-n, closing frits 13-n' ) . For storage and delivery, the columns were closed at the lower tips by caps, about 100 μl washing buffer was filled into each column above the upper frit, and the upper ends were closed by caps as well. Fluorescence Measurements The fluorescence detector Kontron SFM 25 was calibrated with a normalization solution, bovine serum albumin in PBS buffer (0.01 M Na 2 HPO 4 -0.01 M NaH 2 PO 4 -0.15 M NaCl-0.005 % Tween 20-pH 7.2), stabilized with 0.1 % sodium azide. Example 1 Differential Diagnosis of the Early resp. Chronical Phase of Lyme Disease Borrelia burgdorfii, spread out by tick bites in Europe and in the U.S., results in the chronical Lyme Disease (Erythema (chronicum) migrans) . For therapeutical approach and control, the diagnosis of the disease phase is important. This can be done qualitatively (paragraph a) or quantitatively (paragraph b) . a) Qualitative Discrimination between anti-Borellia burgdorfii Antibodies of the Classes IgG and IgM with Integrated Negative Control Field: Reagents: - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0) - Substrate buffer (1 mg/ml PNPP (paranitrophenyl- phosphate) in 1.0 M diethanolamine-0.5 mM MgCl 2 -pH 9.8) - Solution of Borellia b. antigen, isolated from Borellia b. strains, labelled with alkaline phosphatase (10 μg/ml in washing buffer) Column with substrate beds 1-3 3 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg covalently coupled bovine serum albumin (Sigma) 2 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg covalently coupled monoclonal mouse an¬ ti-human IgM antibody (Sigma) 1 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg covalently coupled monoclonal mouse an¬ ti-human IgG antibody (Sigma) Test performance 50 μl of 1:10 dilutions of human serum samples in washing buffer were applied to columns of the described type, followed each by 750 μl washing buffer, by 500 μl of the phosphatase- labelled Borrelia-b. antigen solution, again by 750 μl washing buffer, and finally by 200 μl substrate buffer. After incuba¬ tion until a light yellowish colour appeared in the upper control field 3 (about 10 minutes incubation time for the enzyme reaction, not for the affinity reaction!), both test fields remained colourless (or became slightly yellowish) in the case of negative samples. For positive samples with IgG only, i.e. samples of old diseases, only the lower test fields 1 of the resp. columns developed a significantly strong dark yellow colour, while this happened in both test fields 1 and 2 for samples with both IgG and IgM, i.e. samples of fresh infections. Table 1 Sample Substrate Substrate Substrate Bed 1 Bed 2 Bed 3 Negative Samples 1 - 5 ly - - IgG Positive Samples 6 - 9 iy - dy IgG and IgM Positive Samples 10 - 12 ly dy dy ly = light yellow dy = dark yellow b) Quantitative Determination of anti-Borellia b. IgM and IgG Reagents: - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0) - Elution buffer (0.1 M NaOH-0.15 M NaCl-0.05 % Tween 20-pH 13.0) - Mixed solution of 10 μg/ml mouse anti-human IgG monoclonal antibody, marked with FITC (fluores- ceinisothiocyanate) , and of 10 μg/ml mouse anti- human IgM monoclonal antibody, marked with TRITC (rhodaminisothiocyanate) , in washing buffer (both Sigma) Column with substrate bed 1 1 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg covalently coupled Borrelia b. antigen Test Performance 50 μl of 1:10 dilutions of the human serum samples in washing buffer were applied to columns of the described type, followed each by 750 μl washing buffer, by 500 μl of the mixed antibody solution, and by another 750 μl washing buffer. After elution with 750 μl elution buffer into cuvettes, the relative fluo¬ rescences were determined at 490/520 resp. 555/585 nm. The linear relation between the relative fluorescences and the concentrations of different dilutions of different dilutions of a human serum sample is used as standard line for all further measurements with columns prepared from the same basic stock of antigen-coupled agarose. The standardization of the basic linear line was done by com¬ parison with fluorescence measurements of total IgG resp. IgM amounts retained on antihuman IgG resp. antihuman IgM columns (taking into account the different zero fluorescence data of solutions with no IgG/lgM resp. no anti-Borrelia b. IgG/IgM on the different types of columns) . Example 2 Quantitative Titer of Tetanus and Diphtheria Antibodies as a Measure of the Vaccination Status, i.e. as a Basis for the Decision, whether Vaccination (one or both) is necessary. Tetanus vaccinations are performed in the case of injuries for prophylaxis, mostly. At (already or still) existing sufficient vaccine protection of the patient, the vaccination may cause anaphylactic shock even with fatal outcome. The determination of the vaccination status may as well be important before the application of the combined vaccines for tetanus and diphthe¬ ria (the latter is increasing again in Eastern Europe), i.e. for the decision whether to apply one single vaccine only instead of both. Reagents: - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0) - Elution buffer (0.1 M NaOH-0.15 M NaCl-0.05 % Tween 20-pH 13.0) - mixed solution of 10 μg/ml tetanus toxoids, mar¬ ked with FITC, and of 10 μg/ml diphtheria toxoid, marked with TRITC, in washing buffer (both toxoids were commercial vaccines) - Tetanus standard serum - Diphtheria standard serum (both standards were obtained from Statens Serum Institute, Copenha¬ gen, Denmark, and used as reference for standar¬ dization. ) - Normalization solution for FITC (10 ng/ml fluore- scein in washing buffer) and - Normalization solution for TRITC (10 ng/ml rhoda- mine-in washing buffer) . Column with substrate bed 1: 1 30 μl CnBr-activated Sepharose 4B, loaded with 60 μg covalently coupled Protein G Test Performance The standard sera were diluted in washing buffer to concentra¬ tions of 0.5, 0.4, 0.3, 0.2, and 0.1 IU/ml (IU = international units) (see table 2 and figure 2) . Patient samples from patients of different age were used. The results were compared with results of a commercial ELISA assay (Merlin Diagnostik, Germany) (see table 2) . Exampte2: Quantitative Titer of Tetanus and Diphtheria Antibodies Measurements 1 st Day: FITC- Normalization Solution RFUS = 346 TRITC- Normalization Solution RFUS = 253 Standard RFU RFU NFU NFU lU/ml lU/ml lU/ml lU/ml Solutions FITC TRITC FITC TRITC Tetanus Diphterie Tetanus Diphtheria lU/ml Comparison Comparison ELISA ELISA 0 19 23 0.05 0,09 0 0 0, 1 54 66 0, 16 0,26 0,098 0, 1 1 0,2 89 108 0,26 0,43 0,21 0, 18 0,3 122 153 0,35 0,60 0,32 0,31 0,4 156 197 0,45 0,78 0,39 0,42 0,5 191 243 0,55 0,96 0,53 0,49 Serum Samples No.1 22 87 0,06 0,34 < 0.1 1 ,4 < 0.1 1 ,25 No.2 36 24 0, 10 0.09 0,4 0 0,36 0 Nro3 53 22 0, 17 0,09 1 0 0,94 0 No.4 97 62 0,28 0,25 2,25 0,9 2,36 0.63 No.S 124 34 0,36 0, 13 3, 1 0,25 3,24 0,21 No.6 167 28 0,48 0, 1 1 4,2 0,1 4,05 0.13 No.7 185 45 0,53 0, 18 4,95 0,4 4.75 0,41 No.S 43 22 0, 12 0,09 3,45 0 3,86 0 No. 9 89 28 0,26 0, 1 1 10,2 0,6 9,6 0.56 No. 10 138 25 0,40 0, 10 16,5 0,55 15,3 0,28 Measurement Repetition 2nd Day FITC- Normalization Solution RFUS = 388.00 TRITC- Normalization Solution RFUS = 283,00 Serum Samples No.1 25 97 0.06 0,34 < 0.1 1 ,4 < 0.1 1 ,45 No.S 144 37 0,37 0, 13 3.2 0,24 3,6 0,26 No.9 97 30 0,25 0,1 1 9,8 0,55 9,4 0,52 Measurement Repetition 3rd Day FITC- Normalization Solution RFUS = 329,00 TRITC- Normalization Solution RFUS = 240,00 Serum Samples No.1 21 82 0,06 0,34 < 0.1 1 ,4 < 0.1 1 ,25 No.5 122 31 0,37 0,13 3, 1 0,23 3,3 0,25 No.9 83 26 0,25 0, 1 1 9,9 0,53 9,4 0,56 RFU Relative Flu orescβnce NFU Normalized Fluorescence (RFU / RFUS of Norm alization Soiutio n) IU Internationε .1 Units of Specific Antibodies Sample Dilution No. 1 to Nc . 7 = 1 /10 (s. Test Perfomanoe) Sample Dilution No. 3 to Nc . 10 = 1/SO Table 2 To columns of the described type, the standard solutions resp. 200 μl of the diluted human serum samples (20 μl human serum with 180 μl washing buffer) were applied, followed by 750 μl washing buffer, then by 250 μl of the mixed antigen (toxoid) solution, and at last by 750 μl washing buffer again. After elution with 750 μl elution buffer into cuvettes, the relative fluorescence was determined at 490/520 resp. 555/585 nm. The measurements were repeated twice, one and two days later, with the same precision, using the normalized standard line of the first day. Example 3 a) Quantitative Analysis of (forbidden) Antibiotics in Slaughter Cattle, e.g. of Chloramphenicol, Tetracyclin, and Sulfometazin At the moment, antibiotics in food are mostly determined with bacteriological inhibition tests. These normally take more than 4 days. Therefore, a rapid method is desirable which provides the results even before the slaughter. Reagents: - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0) - Elution buffer (0.1 M NaOH-0.15 M NaCl-0.05 % Tween 20-pH 13.0) - Mixed antibody solution of 10 μg/ml each in wa¬ shing buffer: polyclonal rabbit anti-Chloramphenicol, polyclonal rabbit anti-Tetracyclin, polyclonal rabbit anti-Sulfometacin. (produced by conventional haptene immunization) - Mixed labelled-antigen solution of 10 μg/ml each in washing buffer for qualitative and quantita¬ tive analysis: Chloramphenicol-FITC - Tetracyclin-TRITC and Sulfometacin-Phycocyanine - Normalization solution for FITC (10 ng/ml fluo- rescein in washing buffer) and - Normalization solution for TRITC (10 ng/ml rhoda- mine in washing buffer) . - Normalization solution for phycocyanin (10 ng/ml phycocyanin in washing buffer) . Column with substrate bed 1: 1 30 μl CnBr-activated Sepharose 4B, loaded with 60 μg covalently coupled Protein G Test performance For calibration, 1 ng/ml chloramphenicol, tetracyclin, and sulfometacin are added to bovine serum samples which were proved to be free of these antibiotics by HPLC and by bacteriological inhibition tests. To columns of the described type, 250 μl of the mixed antibody solution were applied. For the calibration and for the demon¬ stration of the enrichment effect on the described columns, to these columns 750 μl of the antibiotics-free sample resp. 750 μl of the prepared antibiotics solution (0.75 ng of each an¬ tibiotic absolute), or 1500, 2250, 3750, or 7500 μl of this solution (1.5, 2.25, 3.75, or 7.5 ng of each antibiotic ab¬ solute) were applied, resp. 750 μl of centrifuged serum sam¬ ples of cattle blood. Then, after 750 μl washing buffer, 250 μl of the mixed fluorescence-labelled antigen solution were added, followed again by 750 μl washing buffer. After elution with 750 μl elution buffer into cuvettes, the fluores¬ cence was determined at 490/520 resp. 555/585 resp. 650/680 nm. The measurements were repeated twice one and two days later, and the same normalized standard line was used (see table 3 and figure 3) . b) qualitative (yes/no) : As shown in table 3, second chapter, last two columns "Confir¬ med", the normalized fluorescence can also just be used for the qualitative evaluation, i.e. whether the sample contains forbidden antibiotics of this group. Rapid qualitative screening was also done using enzyme label¬ ling. The procedure was the same as in a) up to the last wa¬ shing step before the elution, but using a mixed solution of similarly phosphatase-labelled instead of differently fluores¬ cence-labelled antigens. Substrate buffer was then added, followed by an incubation time of 10 minutes. Evaluation was done by comparison of the columns with applied samples to a control column with an applied negative sample. The columns with positive samples all were only slightly yellowish, com¬ pared to the significantly dark yellow colour of columns with negative samples and of the control column. Example 4 Simultaneous Qualitative Test of several Directed IgG and IgM Antibodies for Different Epitopes of one or several Infectants with Integrated Control Fields, especially a Positive Control Field to control Sample Application; exemplified for HIV-I Screening tests for infections (which can be performed simul¬ taneously for several infections just with one column, accor¬ ding to example 2, but just one same label on all different antigens for the antibodies to be tested, i.e. without discri¬ mination between different antibodies) cannot be used for Example 3: Qualitative and Quantitative Determination of Forbidden Antibiotics in Slaughter Cattle Fluorescence of Normalization Solutions Fluorescein RFUS = 346 Rhodamin RFUS = 453 Phycocyanin RFUS = 556 Analyte Cone. RFU RFU RFU NFU NFU NFU Chlora. Tetrac. Sulfom. Chlora. Tetrac. Sulfom. ng absolute 0,00 534 652 765 1,54 1,44 1,38 0,75 497 591 684 1,44 1,30 1,23 1,50 429 524 565 1,24 1,16 1,02 g 2,25 376 471 487 1,09 1,04 0,88 CO 3,75 312 387 . 395 0,90 0,85 0,71 7,50 196 231 219 0,57 0,51 0,39 —i d Serum RFU RFU RFU NFU NFU NFU Confirmed Confirmed Confirmed ri¬ Samples Fπ p¬ s er 1 st Day Chlora. Tetrac. Sulfom. Chlora. Tetrac. Sulfom. Chlora. Tetrac. Sulfom. m (D No.1 546 664 771 1,58 1,47 1,39 - - - m —1 No.2 537 645 762 1,55 1,42 1,37 - - - ) No.3 456 653 773 1,32 1,44 1,39 + - - " 5^ --• No.4 345 661 770 1,00 1,46 1,38 + - - I — rπ No.5 528 545 764 1,53 1,20 1,37 - + - K3 No.6 543 456 769 1,57 1,01 1,38 - + - 3 No.7 531 657 534 1,53 1,45 0,96 - - + No.8 542 649 663 1,57 1,43 1,19 - - + No.9 327 512 769 0,95 1,13 1,38 + + - No.10 478 660 613 1,38 1,46 1,10 + - + 24/1 - Table 3.1 Fluorescence of Normalization Solutions, 2 nd Day Fluorescein RFUS = 374 Rhodamin RFUS = 489 Phycocyanin RFUS = 600 Serum RFU RFU RFU NFU NFU NFU Quantificat. Quantificat. Quantificat. Samples ng/ml ng/ml ng/ml 2nd Day Chlora. Tetrac. Sulfom. Chlora. Tetrac. Sulfom. Chlora. Tetrac. Sulfom. No.1 587 714 826 1,57 1,46 1,38 0 0 0 No.3 480 722 835 1,28 1,48 1,39 1.1 0 0 No.9 349 561 821 0,93 1,15 1,37 3,6 1,5 0 No.10 508 713 648 1,36 1,46 1,08 1,1 0 1,35 Fluorescenc e of Normalization Solutions, 3 rd Day Fluorescein RFUS = 363 Rhodamin RFUS = 480 Phycocyani n RFUS = 587 Serum RFU RFU RFU NFU NFU NFU Quantificat. Quantificat. Quantificat. Samples ng/ml ng/ml ng/ml 3rd Day Chlora. Tetrac. Sulfom. Chlora. Tetrac. Sulfom. Chlora. Tetrac. Sulfom. No.1 563 698 817 1,55 1,45 1,39 0 0 0 31 No.3 473 703 822 1,30 1,46 1,40 1 0 0 No.9 354 551 812 0,98 1,15 1,38 3,2 1.5 0 No.10 504 708 671 1,39 1,48 1.14 1 0 1.3 RFU = Relative Fluorescence NFU = RFU /RFUS of Normalisation Solution Analyte Coi "ic. = Weighted, Added, Absolute Amount of Chloramphenicol, Tetracyclin and Sulfometazin for Standard Curve Chlora Chloramphenicol Tetra Tetracyclin Sulfom Sulfometacin - 26 final decisions. For confirmation and clarification of the positive results, and besides for diagnosis of the actual phase of the diseases, a qualitative or " even quantitative determination of different antibodies is necessary. (The quan¬ titative determination can be performed with one column accor¬ ding to example 2 as well, using different labels for different antigens) . (The confirmation test which is currently used (mainly in blood banks) , Western Blot, has several disadvantages, e.g. there is no integrated control whether the test has been ap¬ plied really (because with negative samples, it remains white like without sample application) , it is a manual test which cannot be automated, it takes much time, and interpretation of the results need much experience) . Reagents : - Washing buffer (0.01 M Tris-0.15 M NaCl-0.05 % Tween 20-pH 8.0) - Substrate buffer (1 mg/ml PNPP in 1.0 M dietha- nolamine-0.5 mM MgCl 2 -pH 9.8) - Mixed antibody solution of 10 μg/ml of mouse an¬ ti-human IgG and 10 μg/ml of mouse anti-human IgM, labelled with alkaline phosphatase, in wa¬ shing buffer Column with substrate beds 1-5 5 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg covalently coupled Bovine Serum Albumin (negative control for non-specific binding) 4 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg HIV-I peptides of the p24 region 3 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg HIV-I peptides of the gp41 region 2 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg HIV-I peptides of the gpl20 region - 27 - 1 30 μl CnBr-activated Sepharose 4b, loaded with 60 μg covalently coupled monoclonal mouse an¬ ti-human IgG (positive control for human serum sample application and sample flow) (antibo¬ dies of Sigma, HIV-I peptides of Saxon Bioche¬ micals, Hannover, Germany) Test performance 500 μl solution (250 μl human serum, diluted with 250 μl wa¬ shing buffer) were applied to columns of the described type, followed by 750 μl washing buffer each, then by 300 μl of the mixed secondary antibody solution, and again 750 μl washing buffer. Then 300 μl substrate buffer were applied, and the columns were incubated for about 6 minutes until the upper negative control field 5 became slightly yellowish (incubation time for enzyme reaction, not for affinity reaction) . Negative control field 5: This field always became slightly yellowish for correctly performed tests and intact columns (test condition) , but dark yellow for an incorrectly stored column (sample 12) . Positive control field 1: After the enzyme incubation time, the positive control field 5 was dark yellow for all human serum samples, i.e. the samples had been applied and flown correctly. With the false rabbit serum sample 11, this field became slightly yellowish only, like the upper control field. Confirmation fields 2-4: In the case of HIV-negative samples 1-5, the test fields 2-4 became slightly yellowish only. All positive samples caused a dark yellow colour in at least one antigen test field. Samples 6 and 7 were from patients with HIV-positive screening tests and negative Western Blot, samples 8-10 were Western Blot confirmed HIV positive. - 28 Table 4 Sample Field 1 Field 2 Field 3 Field 4 Field 5 negative: No. 1 ly iy iy ly dy. No. 2 ly ly iy iy dy No. 3 ly iy iy ly dy No. 4 iy iy iy iy dy No. 5 iy iy iy iy dy questionable: No. 6 iy dy iy iy dy No. 7 iy dy iy iy dy Positive: No. 8 iy dy dy dy dy No. 9 iy dy dy dy dy No. 10 iy dy dy dy dy non-human sample: No. 11 iy iy iy ly ly non-intact column: No. 12 dy dy dy ly ly dy = dark yellow ly = light yellow

Claims: - 29 C l a i m s 1. Reaction column for simultaneous multiple measurement via affinity assay with at least one substrate bed, compri¬ sing at least one affinity reactive component being bound to each substrate bed, and the volume of each bed being up to 600 μl. 2. Reaction column according to claim 1, wherein the sub¬ strate bed is positioned between two porous separating devices. 3. Reaction column according to claim 1 or 2, wherein at least one reactive component is a component for a com¬ pound-specific affinity reaction. 4. Reaction column according to claims 1 to 3, wherein at least one reactive component is a component for a group- specific affinity reaction. 5. Reaction column according to claims 1 to 4, wherein the compound to be analyzed is immunologically reactive. 6. Reaction column according to claims 1 to 5, wherein the substrate bed is a solid porous material or a gel. 7. Reaction column according to claims 1 to 6, wherein the substrate bed and the reactive components for the affini¬ ty reactions are confected and standardized, allowing immediate use of the column without calibration steps or reference measurements. - 30 8. Reaction column according to claims 1 to 7, wherein the reactive component (s) for group-specific affinity reac¬ tions has (have) such a loading density that the com¬ pounds are bound quantitatively to the substrate bed during the controlled flow of the liquid sample before it leaves the column. 9. Reaction column according to claims 1 to 8, wherein the reactive component(s) for compound-specific affinity reactions has/have such a loading density that the com¬ pound(s) is/are bound quantitatively to the substrate bed during the controlled flow of the liquid sample before it leaves the column. 10. Reaction column according to claims 8 and 9, wherein the quantitatively bound compounds are the compounds to be determined, or a group/groups containing them and/or affinity reactive (secondary) binding partners for the compounds all of which are applied to the column in any sensible order and number of steps. 11. Reaction column according to claims 1 to 10, wherein the reactive components are protein A, protein B, protein G, protein M, an immunoglobulin or a group of immunoglobu- lins, an antigen or a group of antigens. 12. Reaction column according to claims 1 to 11, wherein the antigen and the group of antigens include hapten(s) . 13. Reaction column according to claims 1 to 12, wherein the substrate material is selected from the group containing polymeric sugars, plastics, polymer modified organic or inorganic support materials, porous metals, metal oxides, and alloys, glasses, silicates, or ceramics. 31 - 14. Reaction column according to claim 13, wherein agarose is used as polymeric sugar. 15. Reaction column according to claims 1 to 14, wherein the reactive component is bound to the substrate by covalent or any kind of sorptive binding. 16. Reaction column according to claims 2 to 15, wherein porous frits or membranes are used for the upper and/or lower separating devices. 17. Reaction column according to claim 16, wherein the frits have a porosity of 0.2 μm to 100 μm. 18. Reaction column according to claim 16 or 17, wherein the frits are made of plastics, metal or glass. 19. Reaction column according to claim 18, wherein polyethy¬ lene or teflon is used as plastic material respective porous aluminum or stainless steel as metal. 20. Reaction column according to claims 1 to 19, wherein the column material is selected from the group containing plastics, metals, and natural materials. 21. Reaction column according to claim 20, wherein polyethy¬ lene, polypropylene and/or polystyrene are used as pla¬ stic material. 22. Reaction column according to claims 1 to 21, wherein the opposite ends of the reaction columns are shaped for male-female connections for in series connection of se¬ veral reaction columns. 23. Reaction column according to claims 1 to 22, wherein the substrate bed has a volume of 30 to 50 μl. 32 24. Reaction column according to claims 1 to 23, wherein the sequence of substrate beds is arranged in such a manner that a compound which is disturbing the affinity reaction of another compound, is bound quantitatively in an upper substrate bed, while the substrate bed for the affinity reaction of the other compounds takes place in a lower one. 25. Reaction column according to claims 1 to 24, wherein ad¬ ditional beds or zones are integrated above or below the measuring substrate beds, such as filtration devices or zones for sample purification or treatment, additional reaction zones for preparation of the af¬ finity binding reaction, or control zones for control of sample application, sam¬ ple flow, and of applied chemicals. 26. Method for the determination of compounds being determin- able via an affinity reaction, wherein a sample to be analyzed is applied to a reaction column according to claims 1 to 25, and the components to be analyzed or a group/groups containing them are bound quantitatively by the complementary reactive component(s) in the reaction column, whereby the components to be analyzed are deter¬ mined thereafter by determination methods directly in the column, in the liquid directly flown through the column after application or after direct elution or after affi¬ nity assay procedures in the reaction column. 27. Method according to claim 26, wherein (an) affinity reac¬ tive secondary binding partner(s) or (a) (labelled) com¬ petitive component(s) is/are applied to the reaction column before the application of the sample, which satu¬ rate all affinity binding sites in at least one of the substrate beds. 33 - 28. Method according to claims 26 or 27, wherein the affinity assay procedures are secondary respective tertiary affinity reaction(s) in the reaction column for labelling and/or amplification and determination in the column, in the liquid flown through directly after application, or after elution, or affinity reactions of labelled compounds competing with compounds to be determined, and determination directly in the column, directly in the liquid flown through after application, or after elution. 29. Method according to claim 28, wherein fluorescence and/or enzyme labels are used. 30. Method according to claims 26 and 27, wherein the group bound by the group-specific component in the reaction column is the group of immunoglobulins A and/or G and/or M. 31. Method according to claims 26 to 30, wherein similarly labelled complementary antigens or secondary antibodies with one enzyme or fluorescence label for all complemen¬ tary antigens or secondary antibodies is applied for the qualitative determination of the presence of any of spe¬ cial immunoglobulins A and/or G and/or M or of their complementary antigens. 32. Method according to claims 26 to 31, wherein differently labelled antigens or secondary antibodies are applied for quantitative or differentiated qualitative determination of special immunoglobulins A and/or G and/or M, or their corresponding antigens. - 34 - 33. Method according to claim 32, wherein the different anti¬ gen labels are different enzyme and/or fluorescence la¬ bels for each complementary antigen or secondary antibo¬ dy. 34. Method according to claims 26 to 33, wherein control affinity reactions in respective substrate beds are used to control the application and the flow of the sample liquid and the quality of the applied reagents. 35. Method according to claims 26 to 34, wherein the reaction column is directly connected to and remains at the con¬ tainer of the liquid to be tested, or a blood container, for the purpose of safe sampling and test appointment, especially with direct connection to the filling device of the container. 36. Method according to claims 26 to 35, wherein the determi¬ nation is performed without calibration, after one basic calibration of all columns with substrate beds from one basic stock without reference measurements and without regeneration of the substrate bed and of the applied reactive component. 37. Method according to claims 26 to 36, wherein small suc¬ tion or pressure forces are applied to the reaction co¬ lumn after sample application for more rapid performance. 38. Method according to claims 26 to 37, wherein a handling automate is used for automated sampling, sample applica¬ tion, and/or measurement.

Inventors: ERHARDT CHRISTOPH, ERHARDT URSULA, ERHARDT, CHRISTOPH, ERHARDT, URSULA
Applicants: ABION OHG, ERHARDT CHRISTOPH, ERHARDT URSULA, ABION OHG DR. ERHARDT UND MAIER, ERHARDT, CHRISTOPH, ERHARDT, URSULA
Application year: 1994
Main classification: G01N33
IPC codes: B01J20, G01N30, G01N33